Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism required for the addition of CO2 to acetyl-CoA to make malonyl-CoA. In mammals, malonyl-CoA serves as a precursor for the synthesis of fatty acids but it also regulates fatty acid oxidation in mitochondria. There are two ACC isozymes in humans, with very similar amino acid sequences, differing mainly by a 200-residue N-terminal extension on ACC2 that directs this form of the enzyme to mitochondria. There, ACC2-catalyzed synthesis of malonyl- CoA leads to suppression of fatty acid transport and subsequent oxidation. ACC1- synthesized malonyl-CoA is used for fatty acid synthesis. We propose to screen a small- molecule library to find novel inhibitors of ACC2 as lead compounds for the development of new pharmacotherapies to treat obesity. We have constructed yeast gene- replacement strains depending for growth on either human ACC. Our research has already shown that growth of such ACC gene-replacement strains is inhibited by compounds targeting foreign ACCs. We have developed and tested a high throughput protocol necessary to screen large chemical libraries. We also propose to conduct follow-up experiments to determine the specificity of any new inhibitor identified in the screen, using our collection of yeast gene-replacement strains carrying different ACCs, and to confirm ACC as their target, using enzymatic assays. Obesity is a common and prevalent nutritional disorder linked to significant morbidity and premature mortality caused by other serious medical conditions such as diabetes and coronary disease. Acetyl-CoA carboxylase, a key lipid metabolic enzyme, is being investigated as a potential target for new pharmacotherapies to treat the disorder. New medications based on small-molecule inhibitors of the enzyme acting by increasing fatty acid degradation by oxidation or decreasing fatty acid synthesis could become new urgently needed tools to combat the disease. [unreadable] [unreadable] [unreadable]